Project Abstract/Summary Anopheles gambiae mosquitoes, as the insect vector for the Plasmodium parasite that causes malaria, were responsible for the deaths of >450,000 people last year. Fortunately, mosquitoes have a weakness that our research aims to exploit. Mosquitoes use their sense of smell for most human host-seeking behaviors. This suggests that targeting a mosquito's sense of smell could lead to effective measures that prevent bites and the spread of diseases. Indeed, spatial repellents are volatile odorants that effectively disrupt host-seeking behaviors and keep mosquitoes from approaching. They have widespread use in developed nations as personal protective measures, but global adoption is limited due to high costs, unwanted side-effects (such as skin irritation), or the need to use high-concentrations to remain effective. A major limitation to the identification of new, more tractable, repellents is a lack of understanding of how spatial repellents promote repulsion by impacting the mosquito's sense of smell. This is primarily due to challenging technical hurdles needed to link repellent and other odorants to a variety of olfactory neuron functions. Our introduction of the QF2/QUAS genetic binary system into Anopheles mosquitoes overcomes previous technical barriers, and now enables us to directly visualize the response of olfactory neurons, in living mosquitoes, to repellents and human body odors. Using a combination of calcium imaging, single sensillum electrophysiological recordings, and RNA-seq along with our established and novel genetic techniques, we will test the hypothesis that (1) spatial mosquito repellents function by masking, activating, or scrambling the activity of olfactory neurons. We will examine the activity patterns of olfactory neurons in living Anopheles gambiae mosquitoes when stimulated by 20 commonly used mosquito repellents in the presence and absence of human odorants. In addition, we will identify the odorant receptors expressed by olfactory neurons exhibiting altered activity patterns in response to each repellent. Using a combination of genetic approaches (aimed at modulating the function of targeted olfactory neurons) and behavioral assays, we will test the hypothesis that (2) spatial repellents promote repellent behaviors by altering olfactory system signaling. We will experimentally determine which olfactory neurons and what types of olfactory neuron activity changes are either necessary and/or sufficient to drive repellent behaviors. The proposed studies are significant because we will gain new mechanistic insights into how mosquito repellents target the mosquito's sense of smell and enable the development of rationale biology-based strategies to identify new repellents that are cheaper, safer, and more effective for use on a global scale to prevent the spread of disease.